Computational protocol: Controlled electromechanical cell stimulation on-a-chip

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Protocol publication

[…] Device characterization was focused on two aspects, namely membrane deformation as a function of negative pressure (vacuum) and electric signal recording within the cell culture channel.For strain characterization, iron particles (Inframat Advanced Materials, Manchester, CT, USA) with an average diameter of 1 μm were suspended in the PDMS mixture preparation and mixed for 1 min to break up aggregates. In this way, it was possible to embed the particles in the spin-coated membrane and track their displacement (). A range of actuation pressures, ranging from 0 mmHg to −700 mmHg (steps of −50 mmHg), was applied to the pneumatic layer using an eccentric diaphragm vacuum pump (Trivac D&B, TX, USA). Pictures were taken at various actuation conditions using a 200X USB Microscope (AnMo Electronics Corporation, Taiwan) and stored for subsequent image processing.A custom-written MATLAB code (MathWorks, Natick, MA, USA) was used to convert the images in binary format and track 25 bead locations at the vertices of a 5 × 5 matrix, using the particle-tracking algorithm of ImageJ plugin (Speckle TrackerJ). As a result, the X and Y positions of each selected particle spot for each video frame were saved in a dataset file. The dataset files were then analyzed in order to visualize the strain map in different directions (XX, YY, and XY). This procedure was repeated for ten different devices and considering different membrane regions in the same device.For the electrical characterization, a metal needle was injected into the PDMS from the top layer in the center of the cell seeding area, and the signal was recorded with an oscilloscope.After production, each device was checked for mechanical and electrical functionality by controlling leakages during vacuum application and monitoring signals during electrical stimulation. [...] To demonstrate the compatibility of the devices with fluorescent microscopy techniques, cells were stained following standard immunofluorescence protocols. After washing cells with 1x PBS, cells were fixed with 4% paraformaldehyde for 10 min and washed twice in 1x PBS. A solution of 0.1% Triton-X100 was used to permeabilize the cells for 15 min (Image-iT® Fixation⁄Permeabilization Kit, Life Technologies). To prevent non-specific antibody binding, cells were treated for 2 h with a blocking buffer containing 1x PBS, 4% goat serum, and 5% bovine serum albumin. Samples were then incubated overnight at 4 °C with rabbit polyclonal antibody (Abcam, Cambridge, UK) against CX43 at a 1:200 dilution. After three washing steps with washing buffer, WB (1x PBS, 1% bovine serum albumin), cells were incubated with an Alexa Flour® 488 secondary antibody (Life Technologies) at a 1:200 dilution for 1 h in the dark and washed again with WB. Finally, counterstaining was performed using DAPI (Sigma-Aldrich) for nuclei at a 1:200 dilution and ActinRed™ 555 ReadyProbes® Reagent (Life Technologies) for actin cytoskeleton staining. Images were captured using a LSM-780 confocal microscope (Zeiss, Oberkochen, Germany) and processed with Imaris software (Bitplane Scientific Software, Zurich, Switzerland). CX43 expression was quantified from immunofluorescence images through the ImageJ software, for at least three regions of interest (ROIs) for each device. Five devices were considered for each condition. The fluorescence intensities were normalized by the number of cells in the ROIs and plotted as mean ± SD. Cell number per mm2 was assessed by counting the nuclei stained with DAPI and dividing by the area of the ROI. Statistical analysis was performed by a two-tailed t-test. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens